Detection of intracellular RNA or DNA triggers unique sensors to activate the antiviral response: foreign RNA activates the RIG-I-like receptor (RLR)-MAVS pathway, and foreign DNA activates the cGAS-STING pathway. These two antiviral pathways converge on a common set of signaling proteins to activate the type I interferon (IFN) response. However, despite this shared IFN response, the outcomes of RNA and DNA sensing are distinct at both transcriptional and cell biological levels. The unique components of each pathway that are responsible for these distinctions remain largely unknown. We have identified a heat shock protein called HSPA8/HSC70 that is inducibly phosphorylated on a specific serine residue after detection of intracellular DNA, but not after detection of RNA. This is the first signal-dependent, inducible phosphorylation event on HSPA8 identified to date. Interestingly, we have found that DNA-activated phosphorylation of HSPA8 occurs in cells derived from numerous primate and rodent species, but not in mouse cells, strongly suggesting that mice lack this novel innate immune response. HSPA8 plays pleiotropic and essential roles as a protein chaperone and as a regulator of membrane dynamics and autophagy, but its role in the antiviral response is unknown. In this grant, we will define the mechanism and specificity of HSPA8 phosphorylation, we will determine how this phosphorylation impacts the DNA-activated antiviral response, and we will explore the underlying reason for the absence of this response in mouse cells. This project will provide new insights into the DNA-activated antiviral response, it will reveal facets of antiviral immunity that cannot be modeled in mice, and it will create a framework for understanding how heat shock proteins participate in a specific innate immune pathway.